Lactic acid is commonly used as a food additive for preservation, flavor, and acidity. Lactic acid is also used in the manufacture of biodegradable plastic, namely polylactic acid (PLA). The use of PLA as a renewable alternative to petroleum-based products is rapidly expanding (Agrawal, 2003). Physical properties and rate of biological degradation of PLA can be controlled by manipulating the ratio of the chiral substrates, D-lactic acid and L-lactic acid (Narayanan et al., 2004). The global lactic acid market is estimated to be in excess of 100,000 tons per year and is expected to increase substantially in the next few years as new PLA facilities become operational.
For example, demand for the biodegradable solvent ethyl lactate (a derivative of lactic acid) is expected to increase substantially in the near future. It has been estimated that lactate esters could potentially replace as much as 80% of the 3.8 million tons of solvents used each year in the U.S. This solvent is non-toxic and has many useful applications, including in electronic manufacturing, in paints and coatings, in textiles, cleaners and degreasers, adhesives, printing, and de-inking.
Fermentative methods for production of lactic acid are often preferred over chemical synthesis, which results in a mixture of both D and L isomers. The products of microbiological fermentations are dependent on the organism used. Microbiological fermentation can yield a mixture of the two isomers or optically pure lactic acid in a stereospecific form. The desired stereospecificity of the product depends on the intended use.
Bacterial fermentations with Lactobacilli are common for industrial production of lactic acid, but these fermentations rarely yield optically pure product. Additionally, the fastidious nature of these bacteria requires that considerable amounts of supplemental nutrients be added to the growth medium, adding additional cost and making purification more difficult. Moreover, fermentation methods for producing lactic acid are highly inefficient and must be improved to ensure the economic feasibility of the aforementioned anticipated market expansions.
Yeast are not capable of producing appreciable levels of lactic acid, although recombinant Saccharomyces cerevisiae strains have been described that contain the ldh gene from either Lactobacillus or bovine origins (Patent WO 99/14335 and Adachi et al., 1998). While capable of producing up to 2-4% (w/v) lactic acid, these strains exhibit poor productivity and a significant portion of the glucose is converted to ethanol.
The filamentous fungus Rhizopus oryzae (syn. R. arrhizus) is also used for industrial production of lactic acid. R. oryzae is able to aerobically convert glucose, in a chemically defined medium, to large amounts of optically pure L-(+)-lactic acid. Research on lactic acid production by Rhizopus has continued primarily because of the ease of product purification in a minimal growth medium and the ability of the fungus to utilize both complex carbohydrates and pentose sugars (U.S. Pat. No. 4,963,486). This allows the fungus to be utilized for conversion of low value agricultural biomass to lactic acid. Unfortunately, the ability to modify lactic acid production by genetic modification in Rhizopus and other fungi has been limited.
Escherichia coli K-12-based biocatalysts have been engineered for D-(−)-lactate production but were unable to ferment 10% glucose or sucrose to completion in complex or minimal medium (Chang et al., 1999; Dien et al., 2001; Zhou et al., 2003; Zhu and Shimizu 2004).
One of the E. coli biocatalysts, SZ63 (pLOI3501), was developed for sucrose fermentation by functionally expressing the cscR′ cscA′ cscKB′ genes from E. coli B on a plasmid (Shukla et al. 2004). Although capable of efficient fermentation of 5% glucose or sucrose, higher sugar concentrations were incompletely metabolized by this biocatalyst and continuous antibiotic selection was required for plasmid maintenance.
Other biocatalysts derived from E. coli strain B, such as K011 (Deposit No. ATCC 55124), have the native ability to ferment sucrose (Moniruzzaman et al., 1997). As with the strain SZ63, higher sugar concentrations are incompletely metabolized by this biocatalyst and continuous antibiotic selection is required for plasmid maintenance.
Accordingly, a need still exists for improved lactic acid biocatalysts with increased fermentation rates, product titer, and yields to reduce costs associated with the bio-based production of commodity chemicals (Arntzen et al., 1999; Chotani et al., 2000; Datta et al., 1995; Hofvendahl and Hahn-Hagerdal, 2000; and Ohara et al., 2001).